This application relates to reagents for electrochemical test strips such as the type commonly used in detection of blood glucose, and to compositions, test strips and related methods making use of such reagents.
A large fraction of biosensors, measured both by commercial value and academic interest, involve harnessing the specificity of an oxidoreductase enzyme towards a specific molecule (or “analyte”) of interest. The oxidoreductase enzyme in these biosensors is used to catalyze the transfer of electrons off the specific molecule of interest and onto a chemical that is more readily detectable by some transduction mechanism, or vice versa. Transduction mechanisms include measuring concentrations by electrochemical, electrical or optical means. Chemicals that are more readily detectable by these transduction mechanisms are called “mediators” because of their intermediary role between the biological enzyme and the sensing mechanism in the biosensor.
A subset of the oxidoreductase enzymes are the oxidases, which use oxygen already present in a sample as a mediator, often forming hydrogen peroxide as the moiety which has accepted the electrons from the enzyme. Biosensors based on detection of hydrogen peroxide formed an early generation of devices but these were commercially hampered by the difficulty of detecting the peroxide, which is electrochemically detectable only at a catalytic electrode such as platinum.
Several methods to overcome this difficulty were developed. Of some relevance to this invention are the disclosures of Lau et al (U.S. Pat. Nos. 7,135,100 and 7,608,180) where different salts of ferricyanide were used to facilitate transfer of electrons off the hydrogen peroxide (formed when oxygen acts a mediator to regenerate enzyme) and into the electrode. Thus, the ferricyanide in this system does not act as a mediator with respect to the enzyme, but rather acts at one step removed from the enzyme. The salts of Lau are chosen for their solubility in organic solvents and polymers and low solubility in water, which is a criterion aimed at tackling another difficulty with peroxide-mediated biosensors. This is that the analyte of interest is often in a biological sample in the presence of catalase, which decomposes peroxide without release of the electrons. Methods such as the use of membranes had to be developed in biosensors to keep the catalase away from the peroxide generated and membrane-soluble ferricyanides developed so that the signal could reach the electrode and be detected.
Another response to the problems of these peroxide-based biosensors was the development of reagent systems for biosensors based on oxidase enzymes that could operate without the need for oxygen mediation. Such systems commonly used potassium ferricyanide as a direct mediator to restore enzyme to an active state. Salts of ferricyanide other than the potassium salt have been mentioned in combination with oxidoreductase enzymes before and include: ‘sodium ferricyanide’ (as in U.S. Pat. Nos. 7,816,145, 7,749,766, 7,582,123, 7,169,273, 6,258,254) or ‘an alkali metal ferricyanide (e.g., potassium or sodium ferricyanide)’ U.S. Pat. No. 6,187,751. In three other examples, salts of ferricyanide are listed indiscriminately: ‘the metal ion includes, for example, alkali metal ion such as lithium, sodium, and potassium ion; alkaline earth metal ion such as magnesium and calcium ion; and also aluminium and zinc ion’ (Kadota et al U.S. Pat. No. 5,858,695) or targeted towards a solubility in organic solvents (Lau et al U.S. Pat. Nos. 7,135,100 and 7,608,180 mentioned above).
Potassium ferricyanide is a good mediator for a variety of oxidoreductase enzymes. Even for oxidase enzymes such as glucose oxidase, potassium ferricyanide at high concentrations (˜100 mM or greater) is a sufficiently fast mediator that it can mostly outcompete the less concentrated oxygen in a sample, which then becomes only an interferent. The solubility of potassium ferricyanide in water gives another advantage of biosensor formulations that contain it; when ferricyanide is present in large amounts (for example, Walling et al in U.S. Pat. No. 5,508,171 define a useful range for potassium ferricyanide ‘from about 0.15 molar (M) to about 0.7 M’ in their sensor) it can support an adequate counter reaction at the counter electrode to balance the reaction at the working electrode. The counter reaction provides sufficient current and anchors the chemical potential so that no reference electrode is needed nor is it necessary for the counter electrode to be larger than the working electrode. This means sensors can be simplified to two-electrode sensors with electrodes of the same size and material. Other work using this type of reagent is found in WO 97/00441, U.S. Pat. Nos. 5,708,247 and 6,258,229.
However, and perhaps as a consequence of its widespread use, problems with potassium ferricyanide are widely recognized. These include sensitivity to oxygen because even if the ferricyanide can out-compete the oxygen as a mediator, the ferrocyanide produced is still susceptible to oxidation by oxygen. Another problem of ferricyanide is the tendency for some of the ferricyanide to transform into ferrocyanide, even in a dried reagent over a prolonged period, if the potassium ferricyanide is left in intimate contact with the enzyme. Various methods of creating formulations have been disclosed to ameliorate this problem: Nankai et al (U.S. Pat. No. 5,120,420) deposit their enzyme and potassium ferricyanide in layers separated by a ‘hydrophilic high molecular substance layer’ to keep them apart and ensure ‘excellent preservation properties,’ while Walling et al (U.S. Pat. No. 5,508,171) use a microcrystalline cellulose to disperse the potassium ferricyanide.
With the increase in emphasis for faster test times and improved accuracy, the slow mediation rate of potassium ferricyanide (requiring higher concentrations) and its tendency to produce ferrocyanide in the dried reagent mean the ferricyanide ion is falling out of favour as a component in biosensor reagents. To this end, mediators have been developed that do not need such high concentrations to outcompete oxygen e.g. the metal bipyridyl (“bpy”) complexes of U.S. Pat. Nos. 5,378,628, 5,393,903, 5,437,999, 5,410,059, 5,589,326, 5,846,702, metal pyridyl-imidazole complexes of U.S. Pat. Nos. 6,605,201, 6,676,816, 7,074,308 and the bidentate imidazole complexes of U.S. Pat. No. 7,090,756. These types of complex, however do not always have the high solubilities to support the necessary counter reaction and so the strip construction has to be more complex, with the introduction of a reference or Ag/AgCl counter/reference electrode. Types of high-solubility Os complexes have been described in U.S. Pat. No. 5,589,326 but the cost of osmium complexes makes this approach markedly more expensive than potassium ferricyanide for disposable strips.
It is possible to supplement the activity of potassium ferricyanide by adding a second mediator, including those described above; this generally results in the problem of accentuating the worst features of both mediators, in particular: (a) the oxidation potential required must suit the highest mediator oxidation potential, (b) the loss of signal due to oxygen will be dominated by the worst of the two mediators in this respect, and (c) the size of the signal will only be that of the best performing mediator. Furthermore, the addition of a second mediator to a formulation containing potassium ferricyanide accentuates the production of ferrocyanide in the dried reagent. Methods around these problems have to be developed and these include Guo et al's method of physical separation of reagent components on two electrodes in a sandwich (U.S. Pat. No. 6,033,866).
A partial solution to the problem is presented by Harding et al, US 2007/0295616-A1, which is incorporated herein by reference, where careful selection of electrode potentials allows two electron-transfer species to act in concert rather than in parallel. In this system, a mediator regenerates the enzyme, and a shuttle compound serves as the primary or even the only electroactive species for transfer of electrons to and from the electrodes. Specific combinations of mediator and shuttle compounds disclosed in the application include [Os(MeBpy)2(Im)2]2+/3+ or [Os(Mebpy)2Pic]+/2+ as mediators and ferri/ferrocyanide as the shuttle. U.S. Pat. No. 5,508,171, which is incorporated herein by reference, also discloses some systems in which two mediators are employed.